Synthetic peptides for arterial imaging

ABSTRACT

Vascular disease including asymptomatic atherosclerosis can be diagnosed by administering a synthetic peptide or peptide analog having an affinity for, and propensity to accumulate at, a site of vascular injury to a patient, and then detecting the location of the peptide or peptide analog within the patient&#39;s vascular system. The synthetic peptide or peptide analog may include an amino acid sequence sufficiently duplicative of the amino acid sequence of a region of either the apolipoprotein B, apolipoprotein A-I, or elastin proteins such that the peptide or peptide analog accumulates at a site of vascular injury.

The U.S. Government has rights in this invention pursuant to NIH GrantNo. HL32975.

This is a continuation of application Ser. No. 08/201,057, filed Feb.24, 1994, now abandoned; which is a continuation of Ser. No. 08/048,569filed on Apr. 16, 1993, now abandoned; which is a continuation of Ser.No. 07/694,929 filed on May 2, 1991, now abandoned; which is a CIP ofSer. No. 07/518,215 filed on May 3, 1990, now abandoned; which is a CIPof Ser. No. 07/518,142 filed on May 3, 1990, now abandoned; which is aCIP of Ser. No. 07/189,130 filed on May 2, 1988, now abandoned.

BACKGROUND OF THE INVENTION

The invention relates to methods and means useful for the earlydetection of vascular disease, such as atherosclerosis, particularly,methods and means employing labelled synthetic peptides to detectarterial injury.

Atherosclerosis is a disease which causes the thickening and hardeningof the arteries, particularly the larger artery walls. It ischaracterized by lesions or raised fibrous plaques which form within thearterial lumen. The plaques are most prevalent in the abdominal aorta,coronary arteries, or carotid arteries, and they increase progressivelywith age. They commonly present dome-shaped, opaque, glistening surfaceswhich distort the lumen. A lesion typically will consist of a centralcore of lipid and necrotic cell debris, capped by a collagenfibromuscular layer. Complicated lesions will also include calcifieddeposits and exhibit various degrees of necrosis, thrombosis, andulceration.

The injury at, or deformities of, the arterial lumen presented by theplaque and associated deposits result in occluded blood flow, andultimately in angina, cerebral ischemia, renal hypertension, ischemicheart disease, stroke, and diseases of other organs, if untreated. Atpresent, coronary atherosclerosis is still the leading cause of death inthe United States, claiming the lives of over a half million Americansannually, roughly twice as many as are killed by cancer.

Unfortunately, there are no existing diagnostic methods which can detectthe early stages of atherosclerosis and related vascular diseases whichoften are clinically silent. Since lifestyle changes, drug therapy, andother means exist for delaying or reducing vascular occlusion or thestresses on various body organs which result from atheroscleroticlesions, the early detection of atheromatous plaques in the vascularsystem would be of considerable value in permitting preventiveintervention at a time when it can be most effective.

Arteriography, the conventional approach to diagnosing vascular disease,involves catheterization and the injection of radiopaque substances intothe bloodstream in order to image obstructions in the arteries. Thisprocedure involves significant morbidity, in that infection, perforationof the artery, arrhythmia, stroke, infarction, and even death can occur.Because of the risks involved, arteriograms typically are reserved forindividuals with advanced or acute atherosclerotic disease.

A variety of less invasive techniques for the diagnosis of vascularinjury and disease have been proposed. These techniques includeplethysmography, thermography, and ultrasonic scanning (Lees and Myers,Adv. Int. Med. 27:475, 1982).

Other non-invasive approaches to the diagnosis of vascular injury whichhave been proposed by the present inventor involve the administration oflabelled target-seeking biologically active molecules or antibodieswhich preferentially seek out arterial lesions (U.S. patent applicationSer. No. 929,012, entitled "Detection of Vascular Disease", filed Nov.10, 1986) and the administration of labelled low density lipoproteins(LDLs) to the vascular system of a patient (U.S. Pat. Nos. 4,647,445 and4,660,563). LDLs circulating in the blood are known to bind toatherosclerotic plaques (Lees et al., J. Nucl. Med. 24:154, 1983). Thisbinding most likely occurs via apolipoprotein B-100 (apo B-100), theprotein moiety of the LDL molecule, which is responsible for the removalof LDL from the circulation by receptor-mediated uptake in a variety ofcell types. LDLs conjugated to a radioactive label can be used toprovide information on the location and extent of plaque in the vascularsystem by imaging them with a radiation detector. Alternatively, LDLscan be labelled with a non-radioactive, paramagnetic contrast agentcapable of detection in magnetic resonance imaging (MRI) systems.

One disadvantage to this method is that several days are typicallyrequired to isolate LDLs from the patient's blood and to label them.Often, such a delay in diagnosis and subsequent treatment is detrimentalfor critically ill patients. Further, an additional risk of viralinfection is incurred if donor blood is employed as an LDL source.

Consequently, there exists a need for better non-invasive techniques andreagents capable of detecting and mapping early, non-stenosing,non-flow-disturbing atherosclerotic arterial lesions.

Accordingly, it is an object of the present invention to providesynthetic peptides which are useful for detecting and imaging vasculardisease or injury.

It is another object of the invention to provide synthetic peptidesuseful for imaging which are inexpensive and easy to prepare.

It is yet another object of the invention to provide an improved methodof detecting and mapping vascular injury, including vascular injury atits early stages.

Yet another object of the present invention is to provide a method,which is non-invasive, of detecting and mapping vascular injury.

Finally, it is an object of the present invention to provide syntheticpeptides for the prevention or treatment of vascular damage.

SUMMARY OF THE INVENTION

In general, the invention features a peptide or peptide analog having anaffinity for, and propensity to accumulate at, a site of vascularinjury.

By "peptide" is meant any chain of 30 amino acids or less. By "peptideanalog" is meant a peptide which differs in amino acid sequence from thenative peptide only by conservative amino acid substitutions, forexample, substitution of Leu for Val, or Arg for Lys, etc., or by one ormore non-conservative amino acid substitutions, deletions, or insertionslocated at positions which do not destroy the biological activity of thepeptide (in this case, the ability of the peptide to target vascularlesions). A peptide analog, as used herein, may also include, as part orall of its sequence, one or more amino acid analogues, molecules whichmimic the structure of amino acids, and/or natural amino acids found inmolecules other than peptide or peptide analogues.

In preferred embodiments, the peptide or peptide analog is watersoluble; or is soluble in a physiological fluid, preferably, one whichis at physiological pH, for example, blood plasma.

In another preferred embodiment, the peptide has a molecularconformation analogous to the molecular conformation (size, shape,charge) of a surface region of the apolipoprotein B (apo B) moiety ofLDL. Alternatively, the peptide or peptide analog may have an amino acidsequence sufficiently duplicative of at least a portion of a surfaceregion of the apolipoprotein B moiety of LDL, such that the peptide orpeptide analog accumulates at a site of vascular injury in a mannercharacteristic of LDL. Such a surface region is preferably amphiphilic(i.e., having both a hydrophobic and a hydrophilic surface) and is,preferably, also α-helical. Examples of preferred peptide or peptideanalogues include:

Tyr-Arg-Ala-Leu-Val-Asp-Thr-Leu-Lys-Phe-Val-Thr-Gln-Ala-Glu-Gly-Ala-Lys(SEQ ID NO:1);

Tyr-Lys-Leu-Ala-Leu-Glu-Ala-Ala-Arg-Leu-Leu-Ala-Asp-Ala-Glu-Gly-Ala-Lys(SEQ ID NO:2);

Tyr-Lys-Leu-Ala-Leu-Glu-Ala-Ala-Arg-Leu-Leu-Ala-Asn-Ala-Glu-Gly-Ala-Lys(SEQ ID NO:3);

Tyr-Arg-Ala-Leu-Val-Asp-Tyr-Leu-Lys-Phe-Val-Thr-Gln-Leu (SEQ ID NO:4);

Tyr-Arg-Ala-Leu-Val-Asp-Thr-Leu-Lys (SEQ ID NO:5);

Tyr-Ala-Lys-Phe-Arg-Glu-Thr-Leu-Glu-Asp-Thr-Arg-Asp-Arg-Met-Tyr (SEQ IDNO:6);

Tyr-Ala-Ala-Leu-Asp-Leu-Asn-Ala-Val-Ala-Asn-Lys-Ile-Ala-Asp-Phe-Glu-Leu(SEQ ID NO:7);

Tyr-Arg-Ala-Leu-Val-Asp-Thr-Leu-Lys-Phe-Val-Thr-Glu-Gln-Ala-Lys-Gly-Ala(SEQ ID NO:8); and

Tyr-Arg-Ala-Leu-Val-Asp-Thr-Glu-Phe-Lys-Val-Lys-Gln-Glu-Ala-Gly-Ala-Lys(SEQ ID NO:9).

In another preferred embodiment, the peptide or peptide analog includesan amphiphilic domain, preferably including an α-helix, and has a netcharge of -2 or greater, such that the peptide or peptide analogaccumulates at a site of vascular injury. In various preferredembodiments, the peptide or peptide analog is derived from avascular-associated protein. Preferably, the vascular-associated proteinis apolipoprotein A-I (apoA-I) or, more preferably, apolipoprotein B.

By "net charge" is meant the total charge on a peptide at neutral pH andis calculated by adding together the charge (at neutral pH) on each ofthe amino acids of the peptide. By "derived from" is meant having anamino acid sequence identical or substantially identical to the sequenceof, as used herein, a vascular-associated protein. By "substantiallyidentical to" is meant having an amino acid sequence which differs onlyby conservative amino acid substitutions (as described above) or bynon-conservative amino acid substitutions, deletions, or insertionslocated at positions which do not destroy the biological activity of thepeptide (also as described above). By a "vascular-associated protein" ismeant a protein that is naturally associated either with the vascularwall or with an extracellular component of the vascular system,including the proteins elastin and collagen, and carbohydrates such asproteoglycans.

In other preferred embodiments, the peptide or peptide analog has a netcharge of -2 or greater and has an amino acid sequence sufficientlyduplicative of that of at least a portion of an amphiphilic domain ofapolipoprotein A-I such that the peptide or peptide analog accumulatesat sites of vascular injury. A preferred peptide or peptide analog is:

Tyr-Val-Leu-Asp-Glu-Phe-Arg-Glu-Lys-Leu-Asn-Glu-Glu-Leu-Glu-Ala-Leu-Lys-Gln-Lys(SEQ ID NO:10).

In yet another preferred embodiment, the peptide or peptide analogincludes a hydrophobic domain and has a net charge of -2 or greater,such that the peptide or peptide analog accumulates at the site ofvascular injury. Preferably, the peptide or peptide analog is derivedfrom a vascular-associated protein, for example, elastin; the peptide orpeptide analog has an affinity for a vascular wall component, forexample, a collagen, a proteoglycan, or elastin; the peptide or peptideanalog binds elastin with a dissociation constant of 10⁻⁶ or less (i.e.,or with greater affinity, as measured in vitro by the method of Podet etal., Arteriosclerosis and Thrombosis 11:116, 1991); the hydrophobicdomain of the peptide or peptide analog includes a β-strand. In otherpreferred embodiments, the vascular-associated protein is a peptide orpeptide analog having a net charge of -2 or greater and an amino acidsequence sufficiently duplicative of that of at least a portion ofelastin such that the peptide or peptide analog accumulates at sites ofvascular injury. A preferred peptide or peptide analog may include theamino acid sequence:

Tyr-(Val-Gly-Val-Ala-Pro-Gly)_(x) (SEQ ID NO:11),

wherein x is at least 1 and, preferably, 3 (SEQ ID NO:14); or thepeptide or peptide analog may include the amino acid sequence:

Tyr-(Val-Pro-Gly-Val-Gly)_(x) (SEQ ID NO:12),

wherein x is at least 1 and, preferably, 3 (SEQ ID NO:17) or, morepreferably, 4 (SEQ ID NO:13).

In various other preferred embodiments, the peptide or peptide analoghas an acetylated amino terminus and/or an amidated carboxy terminus.Examples of such peptide or peptide analogues include:

NH₂-Tyr-Arg-Ala-Leu-Val-Asp-Thr-Leu-Lys-Phe-Val-Thr-Gln-Ala-Glu-Gly-Ala-Lys-CONH₂(SEQ ID NO:1);

CH₃CONH-Tyr-Arg-Ala-Leu-Val-Asp-Thr-Leu-Lys-Phe-Val-Thr-Gln-Ala-Glu-Gly-Ala-Lys-CONH₂(SEQ ID NO:1);

H₂N-Tyr-Lys-Leu-Ala-Leu-Glu-Ala-Ala-Arg-Leu-Leu-Ala-Asp-Ala-Glu-Gly-Ala-Lys-CONH₂(SEQ ID NO:2);

CH₃CONH-Tyr-Lys-Leu-Ala-Leu-Glu-Ala-Ala-Arg-Leu-Leu-Ala-Asp-Ala-Glu-Gly-Ala-Lys-CONH₂(SEQ ID NO:2);

H₂N-Tyr-Lys-Leu-Ala-Leu-Glu-Ala-Ala-Arg-Leu-Leu-Ala-Asn-Ala-Glu-Gly-Ala-Lys-CONH₂(SEQ ID NO:3);

CH₃CONH-Tyr-Lys-Leu-Ala-Leu-Glu-Ala-Ala-Arg-Leu-Leu-Ala-Asn-Ala-Glu-Gly-Ala-Lys-CONH₂(SEQ ID NO:3)

CH₃ CONH-Tyr-Arg-Ala-Leu-Val-Asp-Tyr-Leu-Lys-Phe-Val-Thr-Gln-Leu-CONH₂(SEQ ID NO:4);

CH₃ CONH-Tyr-Arg-Ala-Leu-Val-Asp-Thr-Leu-Lys-CONH₂ (SEQ ID NO:5);

CH₃CONH-Tyr-Ala-Lys-Phe-Arg-Glu-Thr-Leu-Glu-Asp-Thr-Arg-Asp-Arg-Met-Tyr-CONH.sub.2(SEQ ID NO:6);

H₂N-Tyr-Ala-Ala-Leu-Asp-Leu-Asn-Ala-Val-Ala-Asn-Lys-Ile-Ala-Asp-Phe-Glu-Leu-CONH₂(SEQ ID NO:7);

CH₃CONH-Tyr-Arg-Ala-Leu-Val-Asp-Thr-Leu-Lys-Phe-Val-Thr-Glu-Gln-Ala-Lys-Gly-Ala-CONH₂(SEQ ID NO:8);

CH₃CONH-Tyr-Arg-Ala-Leu-Val-Asp-Thr-Glu-Phe-Lys-Val-Lys-Gln-Glu-Ala-Gly-Ala-Lys-CONH₂(SEQ ID NO:9);

CH₃CONH-Tyr-Val-Leu-Asp-Glu-Phe-Arg-Glu-Lys-Leu-Asn-Glu-Glu-Leu-Glu-Ala-Leu-Lys-Gln-Lys-CONH₂(SEQ ID NO:10);

H₂N-Tyr-Val-Pro-Gly-Val-Gly-Val-Pro-Gly-Val-Gly-Val-Pro-Gly-Val-Gly-Val-Pro-Gly-Val-Gly-CONH₂(SEQ ID NO:13); and

H₂N-Tyr-Val-Gly-Val-Ala-Pro-Gly-Val-Gly-Val-Ala-Pro-Gly-Val-Gly-Val-Ala-Pro-Gly-CONH₂(SEQ ID NO:14).

The synthetic peptide or peptide analogues are useful for detecting andimaging injury in the vascular system of a subject. Other usefulsynthetic peptide or peptide analogues may include: amino acidanalogues, molecules which mimic the structure of amino acids, andnatural amino acids found in molecules other than peptide or peptideanalogues.

In other preferred embodiments, the peptide or peptide analog may belinked to a detectable label to enable its monitoring within thesubject. Preferable labels include a radioisotope, e.g., ¹³¹ I, ¹²⁵ I,¹²³ I, ¹¹¹ In, ^(99m) Tc, ²⁰³ Pb, ¹⁹⁸ Hg, Ru⁹⁷, or ²⁰¹ Tl; or aparamagnetic contrast agent. Such labels may enable the extracorporealmonitoring of synthetic peptide or peptide analogues within the vascularsystem of the subject with, for example, a gamma scintillation camera oran MRI system.

In another aspect, the invention features a method for the detection ofinjury (for example, atherosclerosis) in the vascular system of asubject involving introducing into a subject a peptide or peptide analogof the forms set forth above. The method may further involveadministering a second peptide or peptide analog of the forms set forthabove. The peptide or peptide analog to be introduced may beadministrated by arterial or venous injection. Alternatively, anon-hydrolyzable derivative may be administered orally or nasally. Theintroduced synthetic peptide or peptide analog is then allowed tocirculate within the vascular system of the subject, whereby at least aportion of it accumulates at a site of injury. The portion of thesynthetic peptide or peptide analog which has accumulated at a site ofinjury is then detected. The detection step may further includequantitating the amount of labelled peptide or peptide analog which hasaccumulated at a site of vascular injury; or imaging the region of thesubject's vascular system at which the synthetic peptide or peptideanalog has accumulated, e.g., by extracorporeal monitoring of a peptideor peptide analog having a detectable label (e.g., a radioactive labelor a paramagnetic contrast agent) with a gamma scintillation camera or amagnetic resonance imaging system.

In a final aspect, the invention includes a method for inhibiting thebinding of low density lipoprotein to the vascular wall(s) of a subjectinvolving administering to the subject a therapeutically-effectiveamount of a peptide or peptide analog of the forms set forth above.

Applicants have discovered that vascular diseases, includingasymptomatic atherosclerosis, can be diagnosed by administering asynthetic peptide to a subject, and then detecting the location,pattern, and concentration of the peptide following its accumulation atsites of injury within the subject's vascular system. The techniqueaffords a number of advantages. It is non-invasive; it requires neithercomplex medical equipment, nor highly skilled medical practitioners tobe successfully accomplished; and the peptides used to target vascularlesions may be produced inexpensively, quickly, and in large quantity(e.g., by recombinant DNA technology).

In addition, the peptides of the invention may be used for theprevention or alleviation of vascular diseases such as atherosclerosis.Administration of the peptides of the invention intherapeutically-effective doses can prevent the accumulation of LDL byblocking LDL binding sites.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects of the invention, the various featuresthereof, as well as the invention itself, may be more fully understoodfrom the following description when read together with the accompanyingdrawings in which:

FIG. 1 shows a schematic model of the apo B-100 configuration, whenincluded in the LDL molecule, and surface-exposed regions;

FIG. 2 is a series of helical wheel diagrams indicating the amphiphiliccharacter of representative synthetic peptides;

FIG. 3 is a graphic representation of the binding of anti-SP-4 antibodyto LDL;

FIG. 4 is a graphic representation demonstrating the ability ofanti-SP-4 antibody to bind to SP-4, SP-6, SP-8, and SP-12A;

FIG. 5 shows a representative plasma decay curve for ¹²⁵ I-labelledsynthetic peptide;

FIG. 6 is an onlay autoradiograph of the abdominal aorta of a rabbitwhich has been injected in vivo with ¹²⁵ I-labelled synthetic peptideSP-4, showing labelled healing lesions in the balloon de-endothelializedsection of the aorta (B) as compared with the unlabelled control portion(A).

FIG. 7 shows a photograph (A) and an onlay autoradiograph (B) of theabdominal aorta of a rabbit treated with ¹²⁵ I-labelled syntheticpeptide SP-4, demonstrating that the peptide localizes atatherosclerotic lesions.

FIG. 8 shows a photograph (A) and an onlay autoradiograph (B) of theabdominal aorta of a rabbit treated with ¹²⁵ I-labelled syntheticpeptide, SP-17;

FIG. 9 shows a photograph (A) and an onlay autoradiograph (B) of theabdominal aorta of a rabbit treated with ¹²⁵ I-labelled syntheticpeptide, SP-19a;

FIG. 10 shows a photograph (A) and an onlay autoradiograph (B) of theabdominal aorta of a rabbit treated with ¹²⁵ I-labelled syntheticpeptide, SP-21a;

FIG. 11 shows a photograph (A) and an onlay autoradiograph (B) of theabdominal aorta of a rabbit treated with ¹²⁵ I-labelled syntheticpeptide, SP-28;

FIG. 12 shows a photograph (A) and an onlay autoradiograph (B) of theabdominal aorta of a rabbit treated with ¹²⁵ I-labelled syntheticpeptide, SP-29; and

FIG. 13 shows a photograph (A) and an onlay autoradiograph (B) of theabdominal aorta of a rabbit treated with ¹²⁵ I-labelled syntheticpeptide, SP-30.

DETAILED DESCRIPTION

This invention provides synthetic peptides which have affinity for, andthe propensity to accumulate at, a site of vascular injury, andtherefore are useful in detecting, diagnosing, monitoring, and treatingvascular disease.

Specific examples of such synthetic peptides having thesecharacteristics may have an amino acid sequence that is analogous toportions of known polypeptides which have an affinity for a site ofvascular injury, i.e., have a molecular conformation, charge, and/orsize which is similar to that part of the polypeptide (e.g., low densitylipoprotein or elastin) which is responsible for its affinity forarterial lesions. Alternatively, the synthetic peptides of the presentinvention may be homologous with portions of the apo B-100 moiety ofLDL, the apo A-I moiety of HDL, or elastin.

Design and Synthesis of Peptides

Peptides useful in the invention are those which successfully targetvascular lesions. Thus, it is preferable to fashion such peptides afterthe sequence of a protein which is "vascular-associated", i.e.,naturally associated with a vascular cell surface or with anextracellular component of the vascular system (e.g., proteoglycans,collagen, or elastin). Proteins of this class include: apolipoprotein B(i.e., the protein moiety of low density lipoprotein) and elastin (anatural component of the arterial wall). It is not necessary, and it isoften inconvenient, to use the entire protein molecule (see above).Applicants have discovered that protein fragments can also be used toeffectively target vascular lesions. Examples of useful fragments aredescribed herein. Applicants have shown that such fragments are of lownet charge (i.e., between -2 and +2), allowing an interaction, e.g.,with the highly negatively-charged vascular wall. Applicants have alsoshown that such peptides fall generally into one of two classes: (1)peptides which include an amphiphilic domain, preferably of α-helicalcharacter; and (2) peptides which include a hydrophobic domain (whichfacilitates interaction with a vascular surface or vascular-associatedextracellular component) and a hydrophilic domain of either positivecharge or low negative charge (i.e., -2 or greater; i.e., or morepositive) which facilitates solubility.

Preferred peptides of class I, i.e., those peptides which include anamphiphilic domain (i.e., a domain which has both a hydrophobic and ahydrophilic surface) are identified, e.g., as described in Kaiser andKezdy (Ann. Rev. Biophys. Biophys. Chem. 16: 561, 1987; Science 223:249,1984). Typically, the amphiphilic domain includes a region of secondarystructure, most commonly, an α-helix or a β-strand. Becauseα-helix-containing peptides are generally more soluble thanβ-strand-containing peptides, they are preferred in the invention;increased solubility facilitates in vitro peptide synthesis and peptideadministration to a patient.

Preferred peptides of class II, i.e., those peptides which include both(a) a hydrophobic domain which facilitates interaction with a vascularcell surface or a hydrophobic vascular-associated component (e.g.,elastin) and (b) a positively-charged or slightly negatively-chargeddomain that facilitates solubility are identified using e.g., themethods for predicting hydrophobicity and hydrophilicity describedbelow. Applicants have shown that peptides of this class, even peptidesincluding one or more domains predicted to form β-strands, may beadministered to a subject and used to efficiently target arteriallesions. Peptides of this class likely interact with hydrophobicvascular-associated extracellular components.

The net charge of a peptide is calculated by adding together the chargeson the amino acids of the peptide at neutral pH. The local chargedcharacter (i.e., amphiphilic, hydrophilic, or hydrophobic nature, e.g.,of a region of a peptide) and secondary structure (i.e., the presence ofan α-helix or β-strand) of a particular sequence of amino acids may bepredicted from its primary sequence using any of a number ofmodel-building approaches. For example, to identify an amphiphilicα-helix, one may construct an "Edmundson wheel", and look for thepresence of hydrophobic and hydrophilic residues on opposite faces ofthe resultant cylinder (Schiffer and Edmundson, Biophys. J. 7:121, 1967;hereby incorporated by reference). Alternatively, to identify anamphiphilic, hydrophobic, or hydrophilic domain or a region of secondarystructure, one may use a semi-empirical formula such as the Chou-Fasmanmethod (Chou and Fasman, Ann. Rev. Biochem. 47:251, 1978; herebyincorporated by reference); or the program, PREDICT (based on the GORmethod of secondary structure prediction) (Robson et al., Introductionto Proteins and Protein Engineering; Elsevier, New York, 1986; herebyincorporated by reference). Such a program makes use of the equation:##EQU1## where I(S_(j) =X:X;R_(j+m)) values are derived from astatistical preference for a residue j to be in a conformation X. Thestate of j is evaluated from a summation over m residues of sequence oneither side of j; parameter values are dependent on the identity of theresidue at each position and its contribution to each of the fourstructural types. Values are calculated for each of the states H, E, T,and C; the highest value determines the predicted structure (eitherH=α-helix, E=β-sheet, T=turn, or C=random coil). Finally, amphiphilicitymay be derived from a calculation of the "hydrophobic moment", i.e., themeasure of the amphiphilicity perpendicular to the axis of a periodicpeptide structure; this approach is described in Eisenberg (Ann. Rev.Biochem. 53:595, 1984; hereby incorporated by reference).

It has been shown that it is the charged character (i.e., amphiphilic,hydrophilic, or hydrophobic) and/or secondary structure of a protein,and not its particular amino acid sequence, which facilitates theprotein's interaction with other charged (or hydrophobic) surfaces (see,e.g., Kaiser and Kezdy, Science 223:249, 1984). Accordingly, it ispossible to design any number of peptide analogues, having differentamino acid sequences, provided that the local charge distribution (andoverall net charge) and secondary structure, and hence the biologicalactivity (in this case, the ability to target vascular lesions) ismaintained. Such peptide analogues will generally differ from the nativeprotein sequences by conservative amino acid substitutions (e.g.,substitution of Leu for Val, or Arg for Lys, etc.) well known to thoseskilled in the art of biochemistry. Moreover, peptides may be designedwhich include a region(s) of amphiphilic, hydrophobic, hydrophilicand/or secondary structure embedded within a longer amino acid stretch.Generally, the charged character and secondary structure of such aregion is unaffected by the surrounding amino acid residues. Again, onlythose peptides which are capable of targeting vascular lesions areconsidered to be useful in the invention.

Good candidates for peptides useful in the invention are peptides basedon surface-exposed protein domains (i.e., regions of the protein whichare present on the external surface of a protein molecule, preferably avascular-associated protein molecule) because such regions are mostlikely to interact with the vascular wall or with a vascular-associatedextracellular component. The identity of surface-exposed domains may bedetermined by tryptic digest analysis (see below) and/or by calculationof a region's degree of hydrophobicity/hydrophilicity (e.g., by theChou-Fasman method, Ann. Rev. Biochem. 47:251, 1978); extracellulardomains are generally hydrophilic or amphiphilic in character; suchdomains are frequently surrounded by hydrophobic stretches whichcorrespond to transmembrane domains.

The peptides, once designed, can be synthesized by any of a number ofestablished procedures, including, e.g., the expression of a recombinantDNA encoding that peptide in an appropriate host cell. Alternatively,these peptides can be produced by the established procedure of solidphase peptide synthesis. Briefly, this procedure entails the sequentialassembly of the appropriate amino acids into a peptide of a desiredsequence while the end of the growing peptide is linked to an insolublesupport. Usually, the carboxyl terminus of the peptide is linked to apolymer from which it can be liberated upon treatment with a cleavagereagent. The peptides so synthesized are then labelled with a reagentwhich enables the monitoring of the peptide after its administration toa patient.

Peptides may be tested for their ability to effectively target vascularlesions using an in vivo animal assay (e.g., that assay describedherein). It is known that LDL accumulates both in the balloonde-endothelialized healing arterial wall of the rabbit and in humanatheroma (Roberts et al., J. Lipid Res. 24:1160, 1983; Lees et al., J.Nuclear Med. 24:154, 1983). Accordingly, a rabbit whose abdominal aortahas been balloon de-endothelialized approximately four weeks prior maybe used as a test model for human arterial disease. Other animals orexperimental systems can be used as well, such as Watanabe HeritableHyperlipemic rabbits which have inherited high blood cholesterolsecondary to a deficiency in LDL receptors. This strain of rabbitdevelops spontaneous atherosclerosis at about 2 months of age, and theyoften die of heart attacks.

The rabbit model has been imaged both by onlay autoradiography with ¹²⁵I-labelled LDL and by external imaging with ^(99m) Tc-labelled LDL usinga gamma scintillation camera. In each case, onlay autoradiography of theexcised rabbit aorta has been reliably predictive of the in vivo resultswith extracorporeal imaging. In preparation for vascular administration,each labelled synthetic peptide may be injected in the free state or,alternatively, may be bound to the surface of a lipid emulsion such as acholesterol ester phospholipid microemulsion. The emulsion is theninjected intravenously into the rabbit. Approximately twenty-four hourslater, the rabbit is subjected to extracorporeal monitoring appropriatefor the specific label on the peptide. Alternatively, the rabbit issacrificed, and its aorta removed and washed. The aorta is either cutinto sequential portions which are then monitored in a liquidscintillation counter, or is dried, covered with a layer of polyesterwrap, and placed on a sheet of x-ray film which is then developed toproduce an onlay autoradiogram after suitable storage time in the dark.

Use

The peptides of the invention may be used to diagnose vascular injuryor, alternatively, to inhibit binding of LDL to vascular walls. Ineither case, the peptide is first administered to a subject, e.g., apatient. Administration may be accomplished by arterial or venousinjection. Alternatively a non-hydrolyzable derivative of the peptide(e.g., a keto methylene derivative) may be administered by mouth, oradministration may be accomplished nasally.

In preparation for vascular administration, labelled synthetic peptideis suspended in a pharmaceutically-acceptable carrier (e.g., aphysiological saline solution) or alternatively may be bound to thesurface of a lipid emulsion such as a cholesterol ester phospholipidmicroemulsion (MV), and the emulsion is then injected intravenously.

For diagnostic use, the labelled peptide is administered in an amountsufficient for later detection (generally, 0.5-1 mg intravenously or5-100 mg orally). In preferred embodiments of the invention, the peptideis labelled with, e.g., a radioisotope such as ¹²³ I, ¹²⁵ I or ^(99m)Tc, and peptide accumulation at a site of injury imaged extracorporeallyby radiation detection means such as a gamma scintillation camera;alternatively, the synthetic peptide is labelled with a non-radioactive,paramagnetic contrast agent capable of being detected in MRI systems. Insuch systems, a strong magnetic field is used to align the nuclear spinvectors of the atoms in a patient's body. The field is then disturbedand an image of the patient is read as the nuclei return to theirequilibrium alignments. In the present invention, synthetic peptides canbe linked to paramagnetic contrast agents such as gadolinium, cobalt,nickel, manganese or iron complexes, to form conjugate diagnosticreagents that are imaged extracorporeally with an MRI system.

For treatment of vascular disease (i.e., to inhibit LDL binding tovascular walls), the peptide is administered in atherapeutically-effective dose, generally 5-100 mg intravenously orintramuscularly. Treatment may be repeated, as necessary, to prevent oralleviate vascular damage.

Description of the Preferred Embodiments

There now follows a description of the design and synthesis of samplepeptides useful in the invention. There also follows a description of anin vivo assay used to test the ability of such peptides to targetvascular injury. These examples are provided to illustrate the inventionand should not be construed as limiting.

Apolipoprotein Peptides

Apoliprotein B (apoB) is the protein moiety of low density lipoprotein.The primary structure of apo B-100 has become available by virtue of itscloning (see e.g., Knott et al., Nature 323:734-742, 1986; Yang et al.,Nature 323:738, 1986; Carlsson et al., Nucl. Acids Res. 13:8813, 1985).Further, enzymatic treatment of apo B-100 with trypsin has enabled theidentification of those surface regions which are apparently involved inthe binding of LDL to various cells and tissues (Forgez et al., Biochem.Biophys. Res. Comm. 140:250, 1986; Knott et al., Nature 323:734, 1986).The surface-exposed regions are shown schematically in FIG. 1. The aminoacid sequence analyses of representative tryptic peptides are shown inTABLE 1.

                  TABLE 1*                                                        ______________________________________                                        HPLC                                                                          Fraction                    Corresponding to                                  No.                         Apo B Amino Acid                                  (Tp)   Amino Acid Sequence  Residue Nos..sup.a                                ______________________________________                                        24      (Lys)-Phe-Val-Thr-Gln-Ala-                                                                        1008-1016                                                Glu-Gly-Ala-Lys (SEQ ID NO: 18)                                        123    (Lys)-Leu-Pro-Gln-Gln-Ala                                                                          2091-2106                                                Asn-Asp-Tyr-Leu-Asn-Ser-Phe-                                                  Asn-Asn-Glu-Arg (SEQ ID NO: 19)                                        70     Leu-Pro-Gln-Gln-Ala-Asn-Asp-                                                                       2091-2098                                                Tyr (SEQ ID NO: 20)                                                    49     (Lys)-Phe-Arg-Glu-Thr-Leu-                                                                         2485-2493                                                Glu-Asp-Thr-Arg (SEQ ID NO: 21                                         99     (Arg)-Ile-Ser-Leu-Pro-Asp-                                                                         2679-2685                                                Phe-Arg (SEQ ID NO: 22)                                                161    (Arg)-Thr-Phe-Gln-Ile-Pro                                                                          3218-3236                                                Gly-Tyr-Thr-Val-Pro-Val-Val-                                                  Asn-Val-Glu-Val-Ser-Pro-Phe                                                   (SER ID NO: 23)                                                        134    Tyr-Thr-Val-Pro-Val-Val-Asn-                                                                       3224-3232                                                Val-Glu-Val-Ser-Pro-Phe-Thr-                                                  Ile-Glu-Met-Ser-Ala-Phe-(Gly-                                                 Tyr-Val-Phe-Pro- Lys) (SEQ ID NO: 24)                                  184    (Arg)-Val-Pro-Ser-Tyr-Thr-                                                                         3265-3275                                                Leu-Ile-Leu-Pro-(Ser-Leu-Glu-                                                 Leu-Pro-Val-Leu-His-Val-Pro-                                                  Arg) (SEQ ID NO: 25)                                                   59     (Lys-Ile-Ala-Asp-Phe-Glu-Leu-                                                                      3828-3841                                                Pro-Thr)-Ile-Val-Pro-Glu-                                                     Gln-Thr-Ile-Glu-Ile-Pro-Ser-                                                  ?-Ile (SEQ ID NO: 26)                                                  106    (Arg)-Asn-Leu-Gln-Asn-Asn-                                                                         4080-4094                                                Ala-Glu-Trp-Val-Tyr-Gln-Gly-                                                  Ala-Ile-Arg (SEQ ID NO: 27)                                            ______________________________________                                         .sup.a Residue numbers taken from the complete primary sequence of            apolipoprotein B.                                                             *from Forgez et al., ibid.                                               

Based on the data of Forgez et al. (ibid) the published shed apo Bsequence (described above), and information to those skilled in the artof biochemistry and peptide design (e.g., that described above),synthetic peptides having an amino acid sequence analogous to the aminoacid sequences of surface regions of the apo B moiety of LDL weredesigned. In some cases, the peptides were amidated at their carboxyterminus and/or acetylated at their amino terminus (i.e., the "A" or "a"peptides). Nine representative apo B peptides and their modifiedcounterparts are shown below. The numbers above the amino acid residuesrefer to the primary sequence of apo B.

                       SP-4:                                                                                                                 1008                                  NH.sub.2 -Tyr-Arg-Ala-Leu-Val-Asp-Thr-Leu-Lys-Phe-Val-T                       hr-                                 (SEQ ID NO:1)                                                1016                                                          Gln-Ala-Glu-Gly-Ala-Lys-CONH.sub.2 ;                                       SP-4A:                                                                                                                   1008                               CH.sub.3 CONH-Tyr-Arg-Ala-Leu-Val-Asp-Thr-Leu-Lys-Phe-V                       al-                                 (SEQ ID NO:1)                                                     1016                                                     Thr-Gln-Ala-Glu-Gly-Ala-Lys-CONH.sub.2 ;                                   SP-6:                                                                               1000                                                                    HN.sub.2 -Tyr-Lys-Leu-Ala-Leu-Glu-Ala-Ala-Arg-Leu-Leu                                                             (SEQ ID NO:2)                                1010                                                                          Ala-Asp-Ala-Glu-Gly-Ala-Lys-CONH.sub.2 ;                                   SP-6A:                                                                                    1000                                                              CH.sub.3 CONH-Tyr-Lys-Leu-Ala-Leu-Ala-Leu-Glu-Ala-Ala-A                       rg-Leu-                             (SEQ ID NO:2)                                   1010                                                                    Leu-Ala-Asp-Ala-Glu-Gly-Ala-Lys-CONH.sub.2 ;                                  SP-8:                                                                               1000                                                                    H.sub.2 N-Tyr-Lys-Leu-Ala-Leu-Glu-Ala-Ala-Arg-Leu-Leu-                                                            (SEQ ID NO:3)                                1010                                                                          Ala-Asn-Ala-Glu-Gly-Ala-Lys-CONH.sub.2 ;                                   SP-8A:                                                                                  1000                                                                CH.sub.3 CONH-Tyr-Lys-Leu-Ala-Leu-Glu-Ala-Ala-Arg-Leu-L                       eu                                  (SEQ ID NO:3)                                1010                                                                          Ala-Asn-Ala-Glu-Gly-Ala-Lys-CONH.sub.2 ;                                   SP-12A:                                                                                                                  1008                               CH.sub.3 CONH-Tyr-Arg-Ala-Leu-Val-Asp-Tyr-Leu-Lys-Phe-V                       al                                  (SEQ ID NO:4)                                Thr-Gln-Leu-CONH.sub.2 ;                                                   SP-14A:                                                                       CH.sub.3 CONH-Tyr-Arg-Ala-Leu-Val-Asp-Thr-Leu-Lys-CONH.                       sub.2 ;                             (SEQ ID NO:5)                             SP-15a:                                                                                           2485                                                      CH.sub.3 CONH-Tyr-Ala-Lys-Phe-Arg-Glu-Thr-Leu-Glu-Asp-T                       hr-Arg-                             (SEQ ID NO:6)                                 2495                                                                      Asp-Arg-Met-Tyr-CONH.sub.2 ;                                                  SP-17:                                                                                       3810                                                              H.sub.2 N-Tyr-Ala-Ala-Leu-Asp-Leu-Asn-Ala-Val-Ala-As                       n-Lys-Ile-                          (SEQ ID NO:7)                                 3822                                                                      Ala-Asp-Phe-Glu-Leu-CONH.sub.2 ;                                              SP-19a:                                                                                                                        1008                            CH.sub.3 CONH-Tyr-Arg-Ala-Leu-Val-Asp-Thr-Leu-Lys-Ph                       e-Val-Thr-                          (SEQ ID NO:8)                             Glu-Gln-Ala-Lys-Gly-Ala-CONH.sub.2 ; and                                      SP-21a:                                                                                              1002                                                      CH.sub.3 CONH-Tyr-Arg-Ala-Leu-Val-Asp-Thr-Glu-Phe-Ly                       s-Val-Lys-                          (SEQ ID NO:9)                             Gln-Glu-Ala-Gly-Ala-Lys-CONH.sub.2.                    

Amino acids 2-13 of the apo B peptide, SP-4, were conservativelysubstituted, hydrophobic residue for hydrophobic residue, and chargedresidue for residues having the same charge to produce SP-6 and SP-8.SP-12 is a truncated form of SP-4 in which the last five amino acidresidues were replaced with a single leucine (Leu) residue. SP-14 is atruncated form of SP-12 in which the last five amino acid residues havebeen deleted and the tyrosine (Tyr) residue at position 7 replaced witha threonine (Thr) residue. SP-15a and SP-17 include amino acids2483-2497 (i.e., including Tp 49) and amino acids 3809-3825, (i.e.,including part of Tp 59), respectively. The sequences of SP-19a andSP-21a are variations on the sequence of SP-4. Physical data obtainedfor peptides SP15a, SP17, SP19a, and SP21a are summarized in Table 2.Helical wheel diagrams demonstrating the amphiphilic and α-helicalnature of these peptides are shown in FIG. 2; hydrophobic residues areencircled and charged residues are indicated. Abbreviations are : A,alanine; D, aspartate; E, glutamate; F, phenylalanine; G, glycine; K,lysine; L, leucine; N, asparagine; Q, glutamine; R, arginine; T,threonine; V, valine; Y, tyrosine; Ac, acetyl.

Another amphiphilic peptide (i.e., SP34a) was synthesized based on anapo A-I consensus peptide (termed APOA-I CONSENSUS), i.e., an idealizedα-helix derived from a number of regions of apolipoprotein A-I; thesequence of this consensus peptide is published in Anantharamaiah (Meth.Enzymol. 128:630, 1986). Unlike apolipoprotein A-I, the syntheticpeptide is only weakly charged, and the sequence is preceded by ananimo-terminal tyrosine residue. This peptide was amidated at itscarboxy terminus and acetylated at its amino terminus. This peptide hasa weak net negative charge (i.e., -2; see Table 2).

SP-34a

CH₃CONH-Tyr-Val-Leu-Asp-Glu-Phe-Arg-Glu-Lys-Leu-Asn-Glu-Glu-Leu-Glu-Ala-Leu-Lys-Gln-Lys-CONH₂(SEQ ID NO:10)

Physical data obtained for SP-34a are summarized in Table 2. A helicalwheel diagram of SP-34a is shown in FIG. 2 (described above).

                  TABLE 2                                                         ______________________________________                                                                    Parent Protein                                    Peptide                                                                              Protein  MW.sup.a    <H>.sup.b Charge.sup.c                            ______________________________________                                        SP-15a apo B    2135.0/2135.5                                                                             -0.53     0                                       SP-17  apo B    1950.0/1950.1                                                                             0.26      -1                                      SP-19a apo B    2051.1/2051.0                                                                             0.00      +1                                      SP-21a apo B    2094.1/2094.4                                                                             -0.18     +1                                      SP-34a apo A-I  2535.3/2535.4                                                                             -0.29     -2                                      SP-28  elastin  1622.9/1622.9                                                                             0.62      -1                                      SP-30  elastin  1818.0/1818.1                                                                             0.63      +1                                      SP-29  elastin  1408.8/1408.8                                                                             0.62      +1                                      ______________________________________                                         .sup.a Molecular weight (calculated/observed); expressed as the parent io     (M + H).sup.+, as determined by Fast Atom Bombardment Mass Spectrometry.      .sup.b Mean Hydrophobicity; calculated using the method and scale of          Eisenberg (J. Mol. Biol. 279:125, 1984).                                      .sup.c Charge is expressed as the difference between positively and           negatively charged groups on the peptide at neutral pH.                  

Elastin Peptides

Elastin is a major component of skin, arteries, lung, and other tissues(Rosenbloom; Robert and Robert, Frontiers of Matrix Biology, Vol. 8:Biology and Pathology of Elastic Tissues, 1980; Reddi, ExtracellularMatrix: Structure and Function, 1985). Analysis of various elastinsequences (see Rosenbloom, Meth. Enzymol. 144:172, 1987) indicates thatelastin proteins are generally composed of a number of repeated units.Two such repeated units are the pentapeptide, Val-Pro-Gly-Val-Gly(VPGVG) (SEQ ID NO:15), and the hexapeptide, Val-Gly-Val-ala-Pro-Gly(VGVAPG) (SEQ ID NO:16). Structurally, elastin repeats have been shown,by circular dichroism (Rahman et al., Coll. Czech. Chem. Comm. 52:1356,1987) and by extensive nuclear magnetic resonance studies (Urry et al.,Biopolymers 25:1939, 1986), to contain repeating β-turn structures(Biochem. Biophys. Res. Comm. 153:832, 1988). The hexapeptide haschemotactic properties (Senior et al., J. Cell. Biol. 99:870, 1984).

Elastin-derived peptides useful for targeting arterial lesions include ahydrophobic binding site; this binding site facilitates interaction witha hydrophobic extracellular vascular wall component (e.g., elastin)and/or allows interaction of the peptide with the negatively-chargedvascular wall. To facilitate solubility in physiological fluids, thepeptides preferably include a hydrophilic domain or a net positive orweak negative charge.

Three representative elastin peptides are shown below. SP-28 includesthree repeats of the elastin hexapeptide VGVAPG. SP-30 and SP-29 includefour and three repeats, respectively, of the elastin pentapeptide VPGVG.The SP-30 and SP-29 peptides were amidated at their carboxy terminus.

SP-28

H₂N-Tyr-Val-Gly-Val-Ala-Pro-Gly-Val-Gly-Val-Ala-Pro-Gly-Val-Gly-Val-Ala-Pro-Gly-OH(SEQ ID NO:14);

SP-30

H₂N-Tyr-Val-Pro-Gly-Val-Gly-Val-Pro-Gly-Val-Gly-Val-Pro-Gly-Val-Gly-Val-Pro-Gly-Val-Gly-CONH₂; (SEQ ID NO:13); and

SP-29

H₂N-Tyr-Val-Pro-Gly-Val-Gly-Val-Pro-Gly-Val-Gly-Val-Pro-Gly-Val-Gly-CONH.sub.2.(SEQ ID NO:17).

Physical data obtained for elastin peptides is summarized in Table 2.

Peptide Synthesis and Labelling

Peptides SP-4, SP-4A, SP-6, SP-6A, SP-8, SP-8A, SP-12A, and SP-14A weresynthesized by solid phase peptide synthesis according to theestablished method of Stewart and Young (Solid Phase Peptide Synthesis,2nd ed., pp. 53-123, 1984 The Pierce Chemical Co., Rockford, Ill.,hereby incorporated by reference). These peptides were synthesized usingthe schedule listed in TABLE 2, but any one of the other scheduleslisted in this reference may alternatively be used to generate anydesired peptides (e.g., any peptide described herein).

                  TABLE 2                                                         ______________________________________                                        SCHEDULE FOR SOLID PHASE PEPTIDE SYNTHESIS                                    (Dioxane-HCl Deprotection: DCC* Coupling)                                     Step Reagent     No. Repeats                                                                              Vol (ml)                                                                              Time (min.)                               ______________________________________                                        1    dry CH.sub.2 Cl.sub.2                                                                     4          25      1                                         2a   50% TFA**   1          25      1                                         2b   50% TFA     1          25      20                                        3    dry CH.sub.2 Cl.sub.2                                                                     2          25      1                                         4    dry 2-propanol                                                                            2          25      1                                         5    CH.sub.2 Cl.sub.2                                                                         3          25      1                                         6    5% DIEA°                                                                           1          25      2                                         7    CH.sub.2 Cl.sub.2                                                                         2          25      1                                         8    5% DIEA     1          25      2                                         9    CH.sub.2 Cl.sub.2                                                                         5          25      1                                         10   Introduce   1          20      20                                             symmetric                                                                     anhydride of                                                                  Boc AA°°                                                   11   TFE.sup.# /DIEA/                                                                          1          5       10                                             CH.sub.2 Cl.sub.2 (add)                                                  12   CH.sub.2 Cl.sub.2                                                                         3          25      1                                         13   100% EtOH   3          25      1                                         ______________________________________                                         *dicyclohexylcarbodiimide                                                     **trifluoroacetic acid                                                        °diisopropylethylamine                                                 °°tertbutyloxycarbonyl amino acid                               .sup.# 2,2,2trifluoroethanol                                             

Peptides SP-15a, SP-17, SP-19a, SP-21a, SP-34a, SP28, SP-30 and SP-29were synthesized by manual solid-phase methods using tert-butoxycarbonyl(t-Boc)-based chemistry. (Barany and Merrifield, The Peptides: Analysis,Synthesis, Biology, Academic Press, New York, 1980; Stewart and Young,Solid-Phase Peptide Synthesis, 2nd ed., Pierce Chemical Co., Rockford,Ill., 1984). Sidechain protecting groups for amino acid derivativesincluded: benzyl esters for Asp and Glu, benzyl ethers for Ser and Thr,chlorobenzyloxycarbonyl for Lys, bromobenzyloxy for Tyr, andmesitylenesulfonyl for Arg. The carboxyl terminal amino acid residue wasattached to methylbenzhydrylamine resin with diisopropylcarbodiimide(DIC) by the method of Stewart and Young (supra). The peptide resin waswashed twice with CH₂ Cl₂ and once with 50% TFA in CH₂ Cl₂ /1%dimethylsulfide, and the t-Boc group was removed by treatment for 20minutes with 50% TFA in CH₂ Cl₂ /1% dimethylsulfide, or by treatmentwith 25% TFA in CH₂ Cl₂ /1% dimethylsulfide for 30 minutes. The peptideresin was next washed five times with 5× CH₂ Cl₂ ; neutralized with twowashes of 10% diisopropylethylamine (DIEA) in CH₂ Cl₂ ; and washed fivetimes with CH₂ Cl₂. The next amino acid was coupled by treatment witheither three equivalents of symmetrical anhydride (see below) for 45minutes or four equivalents of active ester (see below) for 2 hours, inthe presence of 1.5 equivalents of DIEA. The peptide resin was thenwashed four times with CH₂ Cl₂ ; twice with 33% ethanol in CH₂ Cl₂ ; andtwice with CH₂ Cl₂.

Symmetrical anhydride-activated amino acids were prepared by treating6.1 equivalents of amino acid with three equivalents of DIC in CH₂ Cl₂for 20 minutes, on ice. Active esters of hydroxybenzotriazole (HOBt)were prepared from four equivalents each of amino acid, HOBt, and DIC indimethyl formamide (DMF) for 30 minutes on ice. Active esters ofethylhydroxyiminocyanoacetate (EACNOx) were prepared from fourequivalents each of amino acid, EACNOc, and DIC in CH₂ Cl₂ for 30minutes, on ice. Completion of coupling at each step was verified by theKaiser ninhydrin test (Kaiser et al., Anal. Biochem. 34:595, 1970).Incomplete couplings were repeated once or twice and, if stillincomplete, the peptide resin was acetylated with acetic anhydride priorto continuation of synthesis. Peptides were deprotected and cleaved fromthe resin either by HF-treatment (performed as directed byImmunodynamics, San Diego, Calif.) or by treatment with 1:10:1:0.5trifluoromethanesulfonic acid:TFA:thioanisole:ethanedithiol by themethod of Yajima et al. (J. Chem. Soc., Chem. Comm. p.107-108, 1974).Crude deprotected peptides were either desalted on a column of SephadexG-25 eluted with 5% acetic acid or were precipitated twice from the TFAsolution with 10 to 100 volumes of ethyl ether. Peptides were thenpurified by reverse-phase HPLC using a Vydac C₁₈ column and a gradientof 0%-90% CH₃ CN/H₂ O containing 0.1% TFA. Solutions of purifiedpeptides were evaporated, redissolved in water, and lyophilized todryness. Identity of peptides was confirmed by Fast Atom BombardmentMass Spectrometric (FAB-MS) analysis.

The synthetic peptides SP-4, SP-4A, SP-6, SP-6A, SP-8, SP-8A, SP-12A,and SP-14A were radiolabelled by the chloramine T method as described inShih et al. (Proc. Natl. Acad. Sci. USA 87:1436, 1990, hereinincorporated by reference). Certain experiments required radiolabelledLDL. In these cases LDL was labelled with ¹²⁵ -iodine by a previouslydescribed modification of the McFarlane iodine monochloride techniquedescribed in Lees et al. (Proc. Natl. Acad. Sci. USA 80:5098, 1983,hereby incorporated by reference). The radiolabelled lipoprotein orsynthetic peptide was separated from unbound radioisotope by passagethrough a gel filtration "desalting" column of Sephadex G-25 or theequivalent.

The synthetic peptides SP-15a, SP-17, SP-19a, SP-21a, SP-34a, SP-28,SP29, and SP30 were radiolabelled with ¹²⁵ I using chloramine-T asfollows.

The peptide (400 μg) was dissolved in 200 μl of 2.5 mM sodiumphosphate/37.5 mM NaCl buffer, pH 7.4 and mixed with 1 mCi (3 μl) ¹²⁵ I.Chloramine-T (30 μl, 8 mg/ml in H₂ O) was added to the mixture and,after 35 seconds, the reaction was quenched by the addition of sodiumbisulfite (60 μl, 8 mg/ml). Radiolabeled peptides were gel-filtered on aBio-Gel P-2 (Bio-Rad, Hercules, Calif.) column (1 cm×30 cm) and elutedwith 0.1% BSA in 0.1M acetic acid. A lead fraction of 5 ml wascollected, followed by 0.45 ml fractions. Iodinated peptide, whicheluted at approximately fractions 9-12, was pooled and the pH adjustedto 5 with 1N NaOH and then to 7.5 with 1M NaHCO₃.

Iodination of SP17 was performed essentially as described above exceptthat the reaction mixture was adjusted to a final concentration of 50%ethanol. Following iodination, the radiolabeled peptide was precipitatedby addition of bovine serum albumin to a final concentration of 10%, andthe precipitate was collected by centrifugation at 2000 rpm for 15minutes. The pellet was then washed four times with 1 ml. (each) ofwater and, after the final wash, the precipitate was dissolved in 5 ml.of 10% BSA. Alternatively, 20% BSA was added to the iodinated peptide(in 50% ethanol) to a final concentration of 10% and a volume notexceeding 5 ml. The solution was then passed through a BioGel P-2 (10cm×1.5 cm) column and eluted with 0.1% BSA in 0.1M acetic acid asdescribed above. Excess buffer was removed using nitrogen pressure, thecolumn was washed with 5 ml of 0.1% BSA/ 0.1M acetic acid, and the mosthighly radioactive fractions pooled for injection.

In an alternative method, the synthetic peptides are labelled eitherdirectly with technetium (Tc), or indirectly through covalent attachmentof a chelating group such as diethylenetriamine pentaacetic acid (DTPA),which is known to chelate a variety of metals including radioisotopessuch as ¹¹¹ -indium.

Direct coupling to ^(99m) Tc is carried out as follows. 50 mCi ^(99m) Tc(in the form of ^(99m) TcO₄ ⁻), in a 5.0 ml aqueous solution, is addedto 1-6 mg, but preferably to 2 mg, synthetic peptide in 0.5 ml of a 0.2M sodium bicarbonate solution, pH 8.0, and mixed thoroughly for 10minutes at room temperature. The pH is raised to 8.0-9.0 if necessarywith 0.25 M sodium hydroxide. To the mixture is then added 10 mg ofreduced sodium dithionite (57.5 mmoles) freshly dissolved in 0.5 mldistilled water. The mixture is gently stirred for 30 minutes at roomtemperature.

The radiolabelled synthetic peptide fraction is separated from uncoupledtechnetium and sodium dithionite by molecular sieve chromatography. A1×50 cm column of Sephadex G-25, equilibrated with a EDTA-bicarbonatebuffer (0.2 M sodium bicarbonate, pH 8.0, 0.001 M EDTA), is suitable forseparation. The column is standardized with blue dextran and potassiumiodide to determine the void volume and the column volume, respectively.The reaction mixture is applied to the column, and bicarbonate-EDTAbuffer is used to elute column fractions. The macromolecular radioactivepeak that elutes at a position characteristic for the synthetic peptideis isolated and ready for use.

Indirect coupling to ^(99m) Tc is carried out as follows. A chelatingligand, e.g., DTPA (as per Hnatowich et al., Science 220:613, 1983) orbromoacetylparaaminobenzyl EDTA (BABE; as per Meares et al., Analyt.Biochem. 142:142, 1984) is covalently bound to the N- or C-terminus ofthe peptide. These references are hereby incorporated by reference.Technetium is then chelated to the DTPA- or BABE-synthetic peptide bythe procedure described above for direct labelling of synthetic peptide.Technetium, in the form of ^(99m) TcO₄ ⁻ is added to the DTPA-syntheticpeptide, and to the mixture is added a solution of reduced sodiumdithionite, pH 8.0-9.0. ^(99m) Tc-labelled synthetic-DTPA peptide isseparated from uncomplexed ^(99m) Tc and sodium dithionite by columnchromatography (as described above). The preparations are thencharacterized by silica gel chromatography essentially as described byMeares et al. (ibid.) and by HPLC. The ^(99m) Tc-labelled peptide isadministered either in a pharmaceutically-acceptable carrier solution orbound to a lipid emulsion.

Structure

To determine whether the molecular conformation or structure of thesynthetic apoB peptides was analogous to the conformation of the apoBmoiety of LDL, a polyclonal antiserum was raised to each peptide and itsability to bind LDL tested. Antiserum raised to human LDL was used asthe control.

Specific anti-LDL antisera may be purchased from a number of sources(e.g., Hoechst Pharmaceutical, Inc., Cincinnati, Ohio and Marburg-Lahn,West Germany; and Hyland Laboratories, Inc., 4501 Colorado Boulevard,Los Angeles, Calif.). Alternatively, antisera may be prepared by anynumber of protocols known to those skilled in the art. For assaysdescribed herein, anti-LDL antiserum was produced as follows. 5-20 mg ofLDL, prepared according to the method of Fischman et al.(Arteriosclerosis 7:361, 1987) in about 1 ml of saline or barbitalbuffer, was emulsified with an equal volume of Freund's completeadjuvant (Difco Laboratories, Detroit, Mich.). This was most easily doneby placing the lipoprotein and the adjuvant in separate 5 ml Luer-Locksyringes with 20-gauge needles and connecting the two needles via a1-inch piece of 0.030 inch inner diameter polyethylene tubing. Thecontents of the syringes are then forcefully expelled from one syringeinto the other several dozen times through the two needles and theconnecting tubing. A stable creamy emulsion was produced which wasfinally passed entirely into one of the syringes, and the connectingtubing is removed. The emulsified antigen was injected subcutaneouslyinto the back of a laboratory rabbit. If several rabbits were to beinjected with the same antigen, larger syringes and larger quantities ofmaterials were used and each rabbit injected with 2 ml of the emulsionrepresenting 1 ml of the original antigen solution. An alternativemethod for preparing emulsion in quantity is to place equal volumes ofantigen solution and adjuvant into one tube of a Mickle disintegrator(Mickle Company, Hampton, Middlesex, England; Brinkmann Instruments,Inc., Westbury, N.Y.) which is stoppered and placed on one of the steelreeds of this magnetic vibrator. A second sample or a water balance isplaced on the other reed, the machine turned on, and the reeds tuned tomaximal excursion for about ten minutes. The resulting emulsion is drawninto syringes through a blunt 18-gauge needle and injectedsubcutaneously through 20-gauge needles.

For the preparation of antisera of high antibody titer the animal may be"boosted" every 3-5 weeks exactly as for the first injection. Goodantiserum is usually obtained after two injections. Animals treated inthis way may be maintained for long periods in the immune state and willyield very large amounts of antiserum. If quantities of antiserum in therange of 1 liter or more are needed, sheep may be used in the samemanner, except that two to three times the amount of immunizing antigenis required. The animals are bled 6-10 days after each boosterinjection. A small test bleeding may be made to check the antibody leveland purity if desired.

The blood is allowed to clot at room temperature for several hours andis then placed overnight in the refrigerator. The samples arecentrifuged in the cold, the clots removed with an applicator stick,recentrifuged to sediment the remaining blood cells, and the serum isdecanted. One milligram per milliliter of sodium azide is added as apreservative. Antisera in constant use may be kept in the refrigerator,or stored at -15° C. to -20 C.

To produce anti-synthetic peptide antisera, purified synthetic peptidewas dissolved in PBS, pH 7.4 at a concentration of 1 mg/ml. The peptidesolution was mixed with an equal volume of complete Freund's adjuvantand vortexed thoroughly until a thick emulsion was produced. New ZealandWhite rabbits (Millbrook Farms, Amherst, Mass.) were injected with atotal of 0.5 mg synthetic peptide administered subcutaneously in thefour dorsal quadrants. The rabbits were given a boost (injected at thesame sites) with 0.5 mg peptide emulsified in incomplete Freund'sadjuvant 2-3 weeks later. Eight to ten days after the first boost, theanimals were given a second, identical boost and were bled of 30 ml 8 to10 days later.

To test for immunological cross-reactivity microtiter plates (Immulon IIDynatech Labs, Chantilly, Va.) were coated with the purified syntheticpeptide or LDL by an overnight incubation at 4° C. with 100 ng peptideper well in 50 mM carbonate, pH 9.6, and blocked for nonspecific bindingwith an additional overnight incubation with phosphate-buffered saline,pH 7.4 (PBS), 1% bovine serum albumin (BSA). Control wells were coatedwith BSA alone. After washing twice with PBS, the wells were filled withserial dilutions (1:10 to 1:100,000 made in PBS, 3% BSA) of a rabbitpolyclonal antibody generated against the synthetic peptide(s) andincubated for 45 minutes at room temperature. Following thorough washing(3× with PBS, 0.1% BSA), the wells were filled with a 1:2000 dilution ofgoat anti-rabbit IgG-horseradish peroxidase conjugate (AtlanticAntibodies, Scarborough, Me.). After a final wash, the wells were filledwith 3,3'-5,5'-tetramethybenzidine (TMB) microwell peroxidase substrate(Kirkegaard and Perry Labs, Gaithersburg, Md.) and read at 650 nm everytwo minutes on an ELISA-5 automated plate reader (Physica, New York,N.Y.). Results (see, e.g., FIGS. 4 and 5) are expressed as the initialvelocity of substrate conversion (change in OD₆₅₀ /hr), which isdetermined by a linear regression of 15 data points per well. Each datapoint represents the average of three measured data points from the sameplate, run at the same time.

FIG. 3 shows ELISA plates coated with LDL and treated with SP-4antiserum. Anti-SP-4 antiserum was able to bind LDL on the plates,providing immunological confirmation that SP-4 and LDL have structuralsimilarities. In analogous experiments, shown in FIG. 4, anti-SP-4antiserum was shown to bind SP-4 and SP-4A as well as the conservativelysubstituted peptides, SP-6, SP-6A, SP-8, SP-8A, SP-12A, and SP-14A,demonstrating that these peptides have structural similarities to SP-4.

Animal Model

The peptides described herein were assayed for their ability to targetsites of vascular injury as follows. Male New Zealand white rabbits (2to 3 kg each) were obtained from ARI Breeding Labs, West Bridgewater,Mass. To induce vascular injury, their abdominal aortas were denuded ofendothelium by a modification of the Baumgartner technique (Fischman etal., Arteriosclerosis 7:361, 1987). Briefly, after each animal wasanesthetized with ketamine and ether or, alternatively, with xylazine(20 mg/ml) and Ketalar (50 mg/ml), the left femoral artery was isolated;a 4F Fogarty embolectomy catheter (Model 12-040-4F, Edwards LaboratoriesIncorporated, Santa Anna, Calif.) was introduced through an arterotomyin the femoral artery and was advanced under fluoroscopic visualizationto the level of the diaphragm. The catheter was inflated to a pressureof about 3 psi above the balloon inflation pressure with radiographiccontrast medium (Conray, Mallinkrodt, St. Louis, Mo.). Three passes weremade through the abdominal aorta with the inflated catheter to removethe aortic endothelium before removal of the catheter, ligation of thefemoral artery, and closure of the wound. The animals were allowed toheal for a period of 4 to 5 weeks before injection of the labelledsynthetic peptides.

Watanabe Heritable Hyperlipemic (WHHL) rabbits were also used as animalmodels. They were obtained from the WHHL Rabbit Program of the NationalHeart Lung and Blood Institute (Bethesda, Md.) at about 3 months of ageand weighing about 1.5 kg. The animals were raised until they were 3-4kg in weight. At this weight, they exhibited marked aorticatherosclerosis.

Each labelled synthetic peptide preparation (containing, for example,150 to 400 or more μCi of ¹²⁵ I-labelled peptide) in column elutionbuffer was injected into the marginal ear vein of the ballooned andhealing New Zealand white rabbits or WHHL rabbits. Serial blood sampleswere obtained from the opposite ear during the ensuing 0-24 hours andwere analyzed for radioactivity. The labelled peptide concentrations inthe blood samples that were withdrawn over the first 10 minutes afterinjection were extrapolated to zero time to determine the time zeroradioactivity in the calculation of average plasma radioactivity. FIG. 5shows a plasma decay curve for the representative ¹²⁵ I-labelledsynthetic peptide SP4. Peptides SP15a, SP17, SP19a, SP21a, SP28, SP34a,and SP30 were cleared rapidly from the plasma with half-lives of aboutone minute or less; after one hour, the plasma levels were less than 10%of the injected dose and fell by an additional 1% over the next threehours. Peptides SP15a, SP17, SP21a, SP28, SP34a, and SP30 leveled off toa plasma level of 3-6%; peptide SP19a cleared more quickly and leveledoff to a concentration of 0.3% (at four hours).

One to twenty-four hours after injection of the labelled syntheticpeptide preparations, each animal was injected intravenously with 4 mlof a 0.5% solution of Evans blue dye (Allied Chemical Company, NationalAniline Division, NY, N.Y.) which stains areas of de-endothelializedaorta blue. After 30 minutes, the animal was sacrificed by a lethalinjection of pentobarbital. After sacrifice, the aorta was removedcompletely, washed in saline, and fixed in 10% trichloroacetic acid.

The washed and fixed aortas from the animals that had been injected withradiolabelled synthetic peptide were opened along the ventral surface.These segments were then pinned out, fixed for 2 hours in 10%trichloroacetic acid, and photographed. The fixed, opened vessels werethen covered with a single layer of plastic (Saran) wrap, placed on highspeed x-ray film (Kodak Orthofilm OH-1), and stored for 3 days to 4weeks in a Kodak "X-Omatic cassette" (24×30 cm) at -70° C. beforedevelopment in a 90 second X-OMAT.

Examples of such autoradiographs are shown in FIGS. 6 and 7 in which the¹²⁵ I-labelled synthetic peptide, SP-4 was used to image arterialinjury. FIG. 6 shows an onlay autoradiograph of a balloonde-endothelialized rabbit showing the labelled, healing lesions in thetest portion of the aorta (B) as compared with the unlabelled controlportion (A). This autoradiograph demonstrates clear-cut localization ofthe synthetic peptide on the image at the healing (re-endothelizing)edge of the aortic lesions produced by the previous trauma. Since thislesion is known to resemble human arteriosclerosis in many importantrespects, including accumulation of lipoproteins and other pathologicalchanges, the ability of the synthetic peptides to localize at the traumasite, and to permit the imaging thereof demonstrates the utility of thepresent invention in imaging vascular disease. FIG. 7A shows aphotograph of stained, naturally occurring lesions in the aorta of aWHHL rabbit, which clearly correspond to the radioactively-labelledlesions shown in the autoradiograph in FIG. 7B.

Similar results were obtained for peptides SP-15a, SP-17, SP-19a,SP-21a, SP-34a, SP-28, and SP-30. Representative results are shown inFIGS. 8-13. De-endothelialized arterial wall is stained blue (with Evansblue dye, as described above) and appears as dark areas in thephotographs; accumulation of radiolabel is indicated by dark areas inthe autoradiographs. All peptides accumulated focally at the leadingedges of regenerating endothelial tissue in a pattern characteristic ofLDL.

These results and others are summarized in TABLE 3 and TABLE 4. Controlpeptides used were SP-2 (part of the heparin and LDL receptor bindingsite of apolipoprotein E) and SP-11A which is a receptor and heparinbinding domain of apolipoprotein B. To compare the relative accumulationof the ¹²⁵ I-labelled synthetic peptides in the aorta and adrenal gland,it was necessary to correct for differences in mean plasma concentrationof the labelled compounds. The mean concentration of syntheticpeptide-associated ¹²⁵ I radioactivity was calculated by numericalintegration of the plasma decay curves and division by the time sinceinjection of the isotope.

                  TABLE 3                                                         ______________________________________                                        Rabbit Compound   Dose            Cir. T.                                                                             Focal                                 ID     Tested     (μCi) Isotope                                                                              (hrs) Acc'm.                                ______________________________________                                        B3     sp4*       410      .sup.125 I                                                                           8     +                                     B85    sp4*       809      .sup.125 I                                                                           4.5   +                                     B100   sp4*       702      .sup.125 I                                                                           5.5   +                                     B87    sp4*       443      .sup.125 I                                                                           (died 10 min.                                                                 p injection)                                B156   sp4*       300.3    .sup.125 I                                                                           6     +                                     B4     sp4* + MV* 80       .sup.125 I                                                                           24    +                                     B39    sp4* + MV°                                                                        456      .sup.125 I                                                                           24    +                                     B38    sp4* + MV°                                                                        516      .sup.125 I                                                                           24    +                                     B44    sp4* + MV°                                                                        1000     .sup.125 I                                                                           24    +                                     B56    sp4* + MV°                                                                        386      .sup.125 I                                                                           24    +                                     B69    sp4* + MV°                                                                        518      .sup.125 I                                                                           1     +                                     B80    sp4* + MV°                                                                        741      .sup.125 I                                                                           24    +                                     B79    sp4* + MV°                                                                        664      .sup.125 I                                                                           4     +                                     B25    sp4* + MV°                                                                        409      .sup.125 I                                                                           24    +                                     B152   sp4* + MV°                                                                        421.2    .sup.125 I                                                                           6     +                                     B24    MV*                 .sup.14 C/.sup.3 H                                                                   24    -                                     B17    MV*                 .sup.14 C/.sup.3 H                                                                   24    -                                     B16    MV*                 .sup.14 C/.sup.3 H                                                                   24    -                                     B90    sp4A*      615      .sup.125 I                                                                           4     +                                     B106   sp4A*      377      .sup.125 I                                                                           5     +                                     B94    sp4A* + MV°                                                                       625      .sup.125 I                                                                           5     +                                     B108   sp4A* + MV°                                                                       382      .sup.125 I                                                                           4     +                                     B59    sp6A* + MV°                                                                       312      .sup.125 I                                                                           24    +                                     B127   sp6A*      350      .sup.125 I                                                                           4     +                                     B122   sp6A*      348      .sup.125 I                                                                           4     +                                     B139   sp6A*      552      .sup.125 I                                                                           4     +                                     B146   sp6A*      550.4    .sup.125 I                                                                           5     +                                     IL-4   sp6A*      282      .sup.125 I                                                                           5     +                                     IL-1   sp6A*      308      .sup.125 I                                                                           5     +                                     B55    sp6*       529      .sup.125 I                                                                           24    +                                     B115   sp6*       418      .sup.125 I                                                                           4     +                                     B115-1 ap6*       398.9    .sup.125 I                                                                           4     +                                     B111   sp8* + MV°                                                                        419      .sup.125 I                                                                           4     +                                     B103   sp8*       424      .sup.125 I                                                                           5     +                                     B124   sp8*       466.2    .sup.125 I                                                                           4     +                                     B124-1 sp8*       455.8    .sup.125 I                                                                           4     +                                     B120   sp8A*      315.3    .sup.125 I                                                                           4     +                                     B120-1 sp8A*      364.5    .sup.125 I                                                                           5     +                                     B137   sp8A*      402.6    .sup.125 I                                                                           4     +                                     B135   sp8A*      427.7    .sup.125 I                                                                           5     +                                     C-14   sp12A*     132.5    .sup.125 I                                                                           4     +                                     C-13   ap12A*     110      .sup.125 I                                                                           4     +                                     B590   sp11       648.1    .sup.125 I                                                                           4     -                                     B690   sp11       363.0    .sup.125 I                                                                           4     -                                     NZW-1  sp12A*     260.0    .sup.125 I                                                                           5     +                                     NZW-2  sp12A*     166.4    .sup.125 I                                                                           4     +                                     NZW-3  sp14A*     941.0    .sup.125 I                                                                           4     +                                     WHHL-1 sp2*       318.9    .sup.125 I                                                                           1     -                                     WHHL-2 sp4*       427.5    .sup.125 I                                                                           1     +                                     WHHL-3 sp4*       487.8    .sup.125 I                                                                           4     +                                     WHHL-4 sp4*       483.5    .sup.125 I                                                                           4     +                                     WHHL-5 sp4*       688.0    .sup.125 I                                                                           4     +                                     WHHL-6 sp11*      126.3    .sup.125 I                                                                           4     -                                     WHHL-7 sp11*      117.1    .sup.125 I                                                                           4     -                                     ______________________________________                                         MV = cholesterol ester microvesicles                                          *= radioactive                                                                °= cold                                                           

                  TABLE 4                                                         ______________________________________                                        Compound.sup.a          Cir. T. Focal                                         Tested    Isotope       (hours) Acc'm                                         ______________________________________                                        SP-34a    .sup.125 I    4       +                                             SP-28     .sup.125 I    4       +                                             SP-30     .sup.125 I    4       +                                             ______________________________________                                         .sup.a All peptides have plasma halflives of approximately one minute.        Radiolabelled peptides were injected intravenously at a dose of between       0.2-1.0 mCi.                                                             

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES:    27                                         - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #         18) LENGTH:                                                         #           amino acid                                                                  (C) STRANDEDNESS:                                                   #        linearOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO - #: 1:                       - Tyr Arg Ala Leu Val Asp Thr Leu Lys Phe Va - #l Thr Gln Ala Glu Gly         #                 15                                                          - Ala Lys                                                                     - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #         18) LENGTH:                                                         #           amino acid                                                                  (C) STRANDEDNESS:                                                   #        linearOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO - #: 2:                       - Tyr Lys Leu Ala Leu Glu Ala Ala Arg Leu Le - #u Ala Asp Ala Glu Gly         #                 15                                                          - Ala Lys                                                                     - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #         18) LENGTH:                                                         #           amino acid                                                                  (C) STRANDEDNESS:                                                   #        linearOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO - #: 3:                       - Tyr Lys Leu Ala Leu Glu Ala Ala Arg Leu Le - #u Ala Asn Ala Glu Gly         #                 15                                                          - Ala Lys                                                                     - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #         14) LENGTH:                                                         #           amino acid                                                                  (C) STRANDEDNESS:                                                   #        linearOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO - #: 4:                       - Tyr Arg Ala Leu Val Asp Tyr Leu Lys Phe Va - #l Thr Gln Leu                 #                 10                                                          - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #         9A) LENGTH:                                                         #           amino acid                                                                  (C) STRANDEDNESS:                                                   #        linearOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO - #: 5:                       - Tyr Arg Ala Leu Val Asp Thr Leu Lys                                                          5                                                            - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #         16) LENGTH:                                                         #           amino acid                                                                  (C) STRANDEDNESS:                                                   #        linearOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO - #: 6:                       - Tyr Ala Lys Phe Arg Glu Thr Leu Glu Asp Th - #r Arg Asp Arg Met Tyr         #                 15                                                          - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #         18) LENGTH:                                                         #           amino acid                                                                  (C) STRANDEDNESS:                                                   #        linearOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO - #: 7:                       - Tyr Ala Ala Leu Asp Leu Asn Ala Val Ala As - #n Lys Ile Ala Asp Phe         #                 15                                                          - Glu Leu                                                                     - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #         18) LENGTH:                                                         #           amino acid                                                                  (C) STRANDEDNESS:                                                   #        linearOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO - #: 8:                       - Tyr Arg Ala Leu Val Asp Thr Leu Lys Phe Va - #l Thr Glu Gln Ala Lys         #                 15                                                          - Gly Ala                                                                     - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #         18) LENGTH:                                                         #           amino acid                                                                  (C) STRANDEDNESS:                                                   #        linearOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO - #: 9:                       - Tyr Arg Ala Leu Val Asp Thr Glu Phe Lys Va - #l Lys Gln Glu Ala Gly         #                 15                                                          - Ala Lys                                                                     - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #         20) LENGTH:                                                         #           amino acid                                                                  (C) STRANDEDNESS:                                                   #        linearOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO - #: 10:                      - Tyr Val Leu Asp Glu Phe Arg Glu Lys Leu As - #n Glu Glu Leu Glu Ala         #                 15                                                          - Leu Lys Gln Lys                                                                         20                                                                - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #         7A) LENGTH:                                                         #           amino acid                                                                  (C) STRANDEDNESS:                                                   #        linearOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO - #: 11:                      - Tyr Val Gly Val Ala Pro Gly                                                                  5                                                            - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #         6A) LENGTH:                                                         #           amino acid                                                                  (C) STRANDEDNESS:                                                   #        linearOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO - #: 12:                      - Tyr Val Pro Gly Val Gly                                                                      5                                                            - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #         21) LENGTH:                                                         #           amino acid                                                                  (C) STRANDEDNESS:                                                   #        linearOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO - #: 13:                      - Tyr Val Pro Gly Val Gly Val Pro Gly Val Gl - #y Val Pro Gly Val Gly         #                 15                                                          - Val Pro Gly Val Gly                                                                     20                                                                - (2) INFORMATION FOR SEQ ID NO:14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #         19) LENGTH:                                                         #           amino acid                                                                  (C) STRANDEDNESS:                                                   #        linearOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO - #: 14:                      - Tyr Val Gly Val Ala Pro Gly Val Gly Val Al - #a Pro Gly Val Gly Val         #                 15                                                          - Ala Pro Gly                                                                 - (2) INFORMATION FOR SEQ ID NO:15:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #         5A) LENGTH:                                                         #           amino acid                                                                  (C) STRANDEDNESS:                                                   #        linearOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO - #: 15:                      - Val Pro Gly Val Gly                                                                          5                                                            - (2) INFORMATION FOR SEQ ID NO:16:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #         6A) LENGTH:                                                         #           amino acid                                                                  (C) STRANDEDNESS:                                                   #        linearOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO - #: 16:                      - Val Gly Val Ala Pro Gly                                                                      5                                                            - (2) INFORMATION FOR SEQ ID NO:17:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #         16) LENGTH:                                                         #           amino acid                                                                  (C) STRANDEDNESS:                                                   #        linearOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO - #: 17:                      - Tyr Val Pro Gly Val Gly Val Pro Gly Val Gl - #y Val Pro Gly Val Gly         #                 15                                                          - (2) INFORMATION FOR SEQ ID NO:18:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #         10) LENGTH:                                                         #           amino acid                                                                  (C) STRANDEDNESS:                                                   #        linearOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO - #: 18:                      - Lys Phe Val Thr Gln Ala Glu Gly Ala Lys                                     #                 10                                                          - (2) INFORMATION FOR SEQ ID NO:19:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #         17) LENGTH:                                                         #           amino acid                                                                  (C) STRANDEDNESS:                                                   #        linearOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO - #: 19:                      - Lys Leu Pro Gln Gln Ala Asn Asp Tyr Leu As - #n Ser Phe Asn Asn Glu         #                 15                                                          - Arg                                                                         - (2) INFORMATION FOR SEQ ID NO:20:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #         8A) LENGTH:                                                         #           amino acid                                                                  (C) STRANDEDNESS:                                                   #        linearOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO - #: 20:                      - Leu Pro Gln Gln Ala Asn Asp Tyr                                                              5                                                            - (2) INFORMATION FOR SEQ ID NO:21:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #         10) LENGTH:                                                         #           amino acid                                                                  (C) STRANDEDNESS:                                                   #        linearOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO - #: 21:                      - Lys Phe Arg Glu Thr Leu Glu Asp Thr Arg                                     #                 10                                                          - (2) INFORMATION FOR SEQ ID NO:22:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #         8A) LENGTH:                                                         #           amino acid                                                                  (C) STRANDEDNESS:                                                   #        linearOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO - #: 22:                      - Arg Ile Ser Leu Pro Asp Phe Arg                                                              5                                                            - (2) INFORMATION FOR SEQ ID NO:23:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #         20) LENGTH:                                                         #           amino acid                                                                  (C) STRANDEDNESS:                                                   #        linearOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO - #: 23:                      - Arg Thr Phe Gln Ile Pro Gly Tyr Thr Val Pr - #o Val Val Asn Val Glu         #                 15                                                          Val Ser Pro Phe                                                                           20                                                                - (2) INFORMATION FOR SEQ ID NO:24:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #         26) LENGTH:                                                         #           amino acid                                                                  (C) STRANDEDNESS:                                                   #        linearOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO - #: 24:                      - Tyr Thr Val Pro Val Val Asn Val Glu Val Se - #r Pro Phe Thr Ile Glu         #                 15                                                          - Met Ser Ala Phe Gly Tyr Val Phe Pro Lys                                     #            25                                                               - (2) INFORMATION FOR SEQ ID NO:25:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #         21) LENGTH:                                                         #           amino acid                                                                  (C) STRANDEDNESS:                                                   #        linearOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO - #: 25:                      - Arg Val Pro Ser Tyr Thr Leu Ile Leu Pro Se - #r Leu Glu Leu Pro Val         #                 15                                                          - Leu His Val Pro Arg                                                                     20                                                                - (2) INFORMATION FOR SEQ ID NO:26:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #         22) LENGTH:                                                         #           amino acid                                                                  (C) STRANDEDNESS:                                                   #        linearOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO - #: 26:                      - Lys Ile Ala Asp Phe Glu Leu Pro Thr Ile Il - #e Val Pro Glu Gln Thr         #                 15                                                          - Ile Glu Ile Pro Ser Ile                                                                 20                                                                - (2) INFORMATION FOR SEQ ID NO:27:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #         16) LENGTH:                                                         #           amino acid                                                                  (C) STRANDEDNESS:                                                   #        linearOPOLOGY:                                                       -     (xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO - #: 27:                      - Arg Asn Leu Gln Asn Asn Ala Glu Trp Val Ty - #r Gln Gly Ala Ile Arg         #                 15                                                          __________________________________________________________________________

We claim:
 1. A diagnostic, synthetic peptide characterized in that ita)bears a detectable label; b) contains thirty or fewer amino acids; c) iswater soluble; d) contains an amphiphilic domain; and e) has an affinityfor, and propensity to accumulate at, a site of vascular injury.
 2. Thediagnostic peptide of claim 1, wherein said peptide is soluble in aphysiological fluid.
 3. The diagnostic peptide of claim 2, wherein saidphysiological fluid is at physiological pH.
 4. The diagnostic peptide ofclaim 3, wherein said physiological fluid is blood plasma.
 5. Thediagnostic peptide of claim 1, said peptide having a molecularconformation analogous to the molecular conformation of a surface regionof the apolipoprotein B (apoB) moiety of low density lipoprotein (LDL),whereby said peptide accumulates at said site.
 6. The diagnostic peptideof claim 5, having an amino acid sequence sufficiently duplicative ofthat of at least a portion of said surface region of apolipoprotein Bmoiety of LDL such that said peptide accumulates at said site in amanner characteristic of low density lipoprotein.
 7. The diagnosticpeptide of claim 1, wherein said amphiphilic surface region furthercomprises an α-helix.
 8. The diagnostic peptide of claim 5, said peptidehaving an amino acid sequence selected from the group consistingofArg-Ala-Leu-Val-Asp-Thr-Leu-Lys-Phe-Val-Thr-Gln-Ala-Glu-Gly-Ala-Lys(amino acids 2-18 of SEQ ID NO: 1);Lys-Leu-Ala-Leu-Glu-Ala-Ala-Arg-Leu-Leu-Ala-Asp-Ala-Glu-Gly-Ala-Lys(amino acids 2-18 of SEQ ID NO: 2);Lys-Leu-Ala-Leu-Glu-Ala-Ala-Arg-Leu-Leu-Ala-Asn-Ala-Glu-Gly-Ala-Lys(amino acids 2-18 of SEQ ID NO: 3);Arg-Ala-Leu-Val-Asp-Tyr-Leu-Lys-Phe-Val-Thr-Gln-Leu (amino acids 2-14 ofSEQ ID NO: 4); Arg-Ala-Leu-Val-Asp-Thr-Leu-Lys (amino acids 2-9 of SEQID NO: 5); Ala-Lys-Phe-Arg-Glu-Thr-Leu-Glu-Asp-Thr-Arg-Asp-Arg-Met-Tyr(amino acids 2-16 of SEQ ID NO: 6);Ala-Ala-Leu-Asp-Leu-Asn-Ala-Val-Ala-Asn-Lys-Ile-Ala-Asp-Phe-Glu-Leu(amino acids 2-18 of SEQ ID NO: 7);Arg-Ala-Leu-Val-Asp-Thr-Leu-Lys-Phe-Val-Thr-Glu-Gln-Ala-Lys-Gly-Ala(amino acids 2-18 of SEQ ID NO: 8); andArg-Ala-Leu-Val-Asp-Thr-Glu-Phe-Lys-Val-Lys-Gln-Glu-Ala-Gly-Ala-Lys(amino acids 2-18 of SEQ ID NO: 9).
 9. The diagnostic peptide of claim 5wherein said amino acid sequencecomprisesArg-Ala-Leu-Val-Asp-Thr-Leu-Lys-Phe-Val-Thr-Gln-Ala-Glu-Gly-Ala-Lys(amino acids 2-18 of SEQ ID NO:1).
 10. The diagnostic peptide of claim 5wherein said amino acid sequencecomprisesLys-Leu-Ala-Leu-Glu-Ala-Ala-Arg-Leu-Leu-Ala-Asp-Ala-Glu-Gly-Ala-Lys(amino acids 2-18 of SEQ ID NO:2).
 11. The diagnostic peptide of claim 5wherein said amino acid sequencecomprisesLys-Leu-Ala-Leu-Glu-Ala-Ala-Arg-Leu-Leu-Ala-Asn-Ala-Glu-Gly-Ala-Lys(amino acids 2-18 of SEQ ID NO:3).
 12. The diagnostic peptide of claim 5wherein said amino acid sequencecomprisesArg-Ala-Leu-Val-Asp-Tyr-Leu-Lys-Phe-Val-Thr-Gln-Leu (aminoacids 2-14 of SEQ ID NO:4).
 13. The diagnostic peptide of claim 5wherein said amino acid sequencecomprisesArg-Ala-Leu-Val-Asp-Thr-Leu-Lys (amino acids 2-9 of SEQ IDNO:5).
 14. The diagnostic peptide of claim 5 wherein said amino acidsequencecomprisesAla-Lys-Phe-Arg-Glu-Thr-Leu-Glu-Asp-Thr-Arg-Asp-Arg-Met-Tyr(amino acids 2-16 of SEQ ID NO:6).
 15. The diagnostic peptide of claim 5wherein said amino acid sequencecomprisesAla-Ala-Leu-Asp-Leu-Asn-Ala-Val-Ala-Asn-Lys-Ile-Ala-Asp-Phe-Glu-Leu(amino acids 2-18 of SEQ ID NO:7).
 16. The diagnostic peptide of claim 5wherein said amino acid sequencecomprisesArg-Ala-Leu-Val-Asp-Thr-Leu-Lys-Phe-Val-Thr-Glu-Gln-Ala-Lys-Gly-Ala(amino acids 2-18 of SEQ ID NO:8).
 17. The diagnostic peptide of claim 5wherein said amino acid sequencecomprisesArg-Ala-Leu-Val-Asp-Thr-Glu-Phe-Lys-Val-Lys-Gln-Glu-Ala-Gly-Ala-Lys(amino acids 2-18 of SEQ ID NO:9).
 18. The diagnostic peptide of claim1, said peptide having a net charge of -2 or greater, whereby saidpeptide accumulates at said site of vascular injury.
 19. The diagnosticpeptide of claim 18, wherein said amphiphilic domain further comprisesan α-helix.
 20. The diagnostic peptide of claim 18, wherein said peptideis derived from a vascular-associated protein.
 21. The diagnosticpeptide of claim, wherein said vascular-associated protein is theapolipoprotein B (apoB) protein.
 22. The diagnostic peptide of claim 20,wherein said vascular-associated protein is the apolipoprotein A-I(apoA-I) protein.
 23. The diagnostic peptide of claim 22, having a netcharge of -2 or greater and an amino acid sequence sufficientlyduplicative of that of at least a portion of said amphiphilic domain ofapoA-I, whereby said peptide accumulates at said site of vascularinjury.
 24. The diagnostic peptide of claim 22, said peptide having anamino acid sequencecomprisingVal-Leu-Asp-Glu-Phe-Arg-Glu-Lys-Leu-Asn-Glu-Glu-Leu-Glu-Ala-Leu-Lys-Gln-Lys(amino acids 2-20 of SEQ ID NO:10).
 25. The diagnostic peptide of claim1, said peptide comprising a hydrophobic domain and having a net chargeof -2 or greater, whereby said peptide accumulates at said site ofvascular injury, and, when said peptide consists of(Val-Gly-Val-Ala-Pro-Gly)_(x) (SEQ ID NO:16), x is at least
 3. 26. Thediagnostic peptide of claim 25, wherein said peptide is derived from avascular-associated protein.
 27. The diagnostic peptide of claim 26,wherein said vascular-associated protein is elastin.
 28. The diagnosticpeptide of claim 25, wherein said peptide has an affinity for a vascularwall component.
 29. The diagnostic peptide of claim 28, wherein saidvascular wall component is a collagen.
 30. The diagnostic peptide ofclaim 28, wherein said vascular wall component is a proteoglycan. 31.The diagnostic peptide of claim 28, wherein said vascular wall componentis elastin.
 32. The diagnostic peptide of claim 31, wherein said peptidebinds elastin with a dissociation constant of 10⁻⁶ or less.
 33. Thediagnostic peptide of claim 6 or 25, wherein said hydrophobic domaincomprises a β-strand.
 34. The diagnostic peptide of claim 27, having anet charge of -2 or greater and an amino acid sequence sufficientlyduplicative of that of at least a portion of elastin, whereby saidpeptide accumulates at said site.
 35. The diagnostic peptide of claim27, said peptide having an amino acid sequencecomprising(Val-Gly-Val-Ala-Pro-Gly)_(x) (SEQ ID NO: 16), wherein x is atleast
 3. 36. The diagnostic peptide of claim 35, wherein said x is 3(amino acids 2-19 of SEQ ID NO:14).
 37. The diagnostic peptide of claim27, wherein said amino acid sequence comprises(Val-Pro-Gly-Val-Gly)_(x)(SEQ ID NO: 15), wherein x is at least
 1. 38. The diagnostic peptide ofclaim 37, wherein said x is 4 (amino acids 2-21 of SEQ ID NO:13). 39.The diagnostic peptide of claim 37, wherein said x is 3 (amino acids2-16 of SEQ ID NO: 17).
 40. The diagnostic peptide of claim 1, 5, 18, or25, wherein the amino terminus of said peptide is acetylated.
 41. Thediagnostic peptide of claim 1, 5, 18, or 25, wherein the carboxylterminus of said peptide is amidated.
 42. The diagnostic peptide ofclaim 1, wherein said label is radioactive.
 43. The diagnostic peptideof claim 41, wherein said radioactive label is selected from the groupconsisting of ¹³¹ I, ¹²⁵ I, ¹²³ I, ¹¹¹ In, ^(99m) Tc, ²⁰³ Pb, ¹⁹⁸ Hg, ⁹⁷Ru, and ²⁰¹ Tl.
 44. The diagnostic peptide of claim 43, wherein saidradioactive label is ^(99m) Tc.
 45. The diagnostic peptide of claim 1,wherein said label comprises a paramagnetic contrast agent.
 46. Thediagnostic peptide of claim 1, wherein said amphiphilic surface regionfurther comprises a β-strand.